Heavy Metal Resistant, Alkalitolerant Bacteria Isolated From Serpentinizing Springs in the Zambales Ophiolite, Philippines

Serpentinization involves hydrologic alteration of ultramafic mantle rocks containing olivine and pyroxene to produce serpentine minerals. The fluids resulting from this reaction are reduced, extremely depleted in dissolved inorganic carbon, and are highly alkaline with pH values typically exceeding 10. Major byproducts of the serpentinizing reaction include iron oxides, hydrogen, methane, and small amounts of organic molecules that provide chemosynthetic energy for subsurface microbial communities. In addition, weathering of serpentine rocks often produces fluids and sediments that have elevated concentrations of various toxic heavy metals such as chromium, nickel, cobalt, copper, and zinc. Thus, microorganisms inhabiting these unique ecological niches must be adapted to a variety of physicochemical extremes. The purpose of this study is to isolate bacteria that are capable of withstanding extremely high concentrations of multiple heavy metals from serpentine fluid-associated sediments. Fluid and sediment samples for microbial culturing were collected from Manleluag Spring National Park located on the island of Luzon, Philippines. The area is part of the Zambales ophiolite range, and hosts several serpentinizing fluid seeps. Fluid emanating from the source pool of the spring, designated Manleluag 2 (ML2), has a pH of 10.83 and temperature of 34.4 °C. Luria-Bertani agar medium was supplemented with varying concentrations of five trace elements - Cu, Cr, Co, Ni, and Zn. Environmental samples were spread on each of these media and colony forming units were subsequently chosen for isolation. In all, over 20 isolates were obtained from media with concentrations ranging from 25 mg/L - 400 mg/L of each metal. Taxonomic identity of each isolate was determined using 16S rRNA gene sequences. The isolates were then tested for tolerance to alkaline conditions by altering LB medium to pH values of 8, 9, 10, 11, and 12. The majority of strains exhibit growth at the highest pH tested, demonstrating that alkalitolerant, highly metal resistant organisms are found in this serpentinizing system. These organisms are of great interest as they may be exploited for bioremediation, enzyme production, and other biotechnological applications.